The immune response comprises a cellular response and a humoral response. The cellular response is mediated largely by T lymphocytes (alternatively and equivalently referred to herein as T-cells), while the humoral response is mediated by B lymphocytes (alternatively and equivalently referred to herein as B-cells).
B-cells produce and secrete antibodies in response to the presentation of antigen and MHC class II molecules on the surface of antigen presenting cells. Antigen presentation initiates B-cell activation with the engagement of the B-cell receptor (BCR) at the cell's surface. Following engagement, the BCR relays signals that are propagated through the cell's interior via signal transduction pathways. These signals lead to changes in B-cell gene expression and physiology, which underlie B-cell activation.
T-cells produce costimulatory molecules, including cytokines, that augment antibody production by B-cells during the humoral immune response. Cytokines also play a role in modulating the activity of T-cells themselves. Many T-cells act directly to engulf and destroy cells or agents that they recognize by virtue of the cell surface proteins they possess. The engagement of cell surface receptors on T-cells results in the propagation of intracellular signals that provoke changes in T-cell gene expression and physiology, which underlie the cellular immune response.
Antigen recognition alone is usually not sufficient to initiate a complete effector T or B-cell response. The generation of many B-cell responses to antigen is dependent upon the interaction of B-cells with CD4+ helper T-cells directed against the same antigen. These helper T-cells express CD40L (CD154) which binds to the cell surface receptor, CD40, on resting B-cells. This interaction provides a critical activation signal to B-cells. Mutations in the CD40L lead to the X-linked immunodeficiency disorder hyper-IgM syndrome, which is characterized by low levels of IgA and IgG, normal to elevated levels of IgM, absence of germinal center formation, and decreased immune response. In addition, transgenic mice lacking CD40 exhibit reduced graft rejection. (Zanelli et al., Nature Medicine, 6: 629-630, 2000; Schonbeck et al., Cell Mol Life Sci, 58:4-43, 2001).
Non-lymphocyte leukocytes and platelets are also activated by surface receptor engagement in immune response and in response to injury. For example, mast cells and basophils are activated by binding of antigen to surface IgE, while platelets are activated by the binding of thrombin to its receptor.
Intercellular communication between different types of lymphocytes, as well as between lymphocytes and non-lymphocytes in the normally functioning immune system is well known. Much of this communication is mediated by cytokines and their cognate receptors. Cytokine-induced signals begin at the cell surface with a cytokine receptor and are transmitted intracellularly via signal transduction pathways. Many types of cells produce cytokines, and cytokines can induce a variety of responses in a variety of cell types, including leukocytes. The response to a cytokine can be context-dependent as well as cell type specific.
Dysregulation of intercellular communication can perturb leukocyte activity and the regulation of immune responses. Such dysregulation is believed to underlie certain autoimmune disease states, hyper-immune states, and immune-compromised states. Such dysfunction may be cell autonomous or non-cell autonomous with respect to lymphocytes.
The activation of specific signaling pathways in leukocytes determines the quality, magnitude, and duration of immune responses. In response to transplantation, in acute and chronic inflammatory diseases, and in autoimmune responses, it is these pathways that are responsible for the induction, maintenance and exacerbation of undesirable leukocyte responses. Identification of these signaling pathways is desirable in order to provide diagnostic and prognostic tools, as well as therapeutic targets for modulating leukocyte function in a variety of disorders or abnormal physiological states. In addition, the ability to modulate these pathways and suppress normal immune responses is often desirable, for example in hosts receiving a transplant.
A number of signals are known to regulate cellular functions by modulating the flow of cations through cell membranes. The flow of ions through the lipid bilayers of membranes is effected by ion channels. Ion channels are fundamental to cellular functions such as transmission of signals in the nervous system, cell division, and the production of antibodies by lymphocytes.
The forces that influence the movement of ions through a channel are electrical and chemical. The electrical force is the electrical potential across the membrane, the chemical force is the difference in concentration of an ion on the two sides of the membrane: the combination of the two is the electrochemical gradient for an ion. If the electrochemical gradient for an ion is not zero, ions will flow through a channel when it opens if the channel is permeant to that ion.
There are many varieties of ion channels that differ in their selectivity, methods of gating, conductance and kinetic properties. Some channels are selectively permeant to particular cations, including sodium, potassium, and calcium. Other are selective for particular anions, such as chloride. Channels are classified according to the ions that pass through them most freely. For example, sodium channels are more permeable to sodium than to any other cations or anions.
Channels are also classified according to the way in which their open/closed conformations are regulated, i.e., how they are gated. For example, voltage-activated channels open or close in response to changes in membrane potential. Some receptors are ligand-gated channels and are opened when ligands such as neurotransmitters bind to their surface. Other channels are indirectly linked to receptors by second messenger systems, which regulate channel opening and closing.
Channels can also have very different conductances. Conductance, the reciprocal of resistance, is a measure of the ease with which ions pass through a channel and is given by the ratio of the current to the driving force. The conductance of different channels can range from picosiemens to hundreds of picosiemens (corresponding to resistances of 109 to 1012 ohms). Finally, channels can have very different “duty cycles”. Some are open most of the time while others open infrequently. Some flicker rapidly between open and closed states while others do not. Changes in the environment of channels (e.g., the presence of drugs) can change these characteristics. Indeed it is becoming clear that many drugs exert their effects on cells and organs by binding to surface receptors and influencing channel behaviour.
Ion channels are useful pharmacological targets. A number of currently used pharmaceuticals act on ion channels. Calcium channel blockers are used as anti-angina and antihypertensive agents. Barbiturates cause sleep and inhibit epileptic seizures by increasing the movement of chloride ions through gamma-amininobutyric acid (GABA)-activated channels. Similarly, benzodiazepines relieve anxiety and produce anaesthesia by increasing GABA receptor activity and chloride ion conductance. Lymphocytes express a variety of cation channels, including potassium and calcium ion channels. A prolonged rise in intracellular calcium is required for lymphocyte activation, and the blockade of voltage-gated potassium channels and calcium-sensitive potassium channels inhibits antigen-induced activation of lymphocytes, likely by inducing membrane depolarization and thereby diminishing calcium influx (Lewis et al., Ann. Rev. Immunol., 13:623-653, 1995).
One manner by which intracellular calcium regulates lymphocyte activation is through the regulation of calcineurin activity. Calcineurin is a calcium-sensitive protein serine/threonine phosphatase comprised of a catalytic and a regulatory subunit. Calcineurin is activated by the binding of Ca2+/calmodulin, and is inhibited by the immunosuppressants FK506 and cyclosporin A. Binding of cyclosporin to calcineurin blocks substrate access to the active site of the phosphatase (Liu et al., Cell, 66: 807, 1991).
The activation of calcineurin is an important regulatory step in lymphocyte activation. Calcineurin regulates phosphorylation of the nuclear factor in activated T-cells (NFAT) transcription factor, and thereby regulates nuclear import of NFAT and its ability to regulate transcription. The expression of many factors involved in lymphocyte activation, including cytokines (such as IL-2) and cell surface molecules (eg. CD40L) lies downstream of NFAT activation. (see Klee et al., J Biol Chem., 273:13367, 1998; Stankunas et al., Cold Spring Harbor Symposia Quant. Biol., 64: 505-516, 1999).
The identification of cation channels associated with lymphocyte activation is desirable for the development of therapeutics that modulate the activity of such channels, which may be used for the treatment of autoimmune disorders, acute and chronic inflammatory disorders, and the management of immune responses in a host receiving a graft.